MdNup62 involved in salt and osmotic stress tolerance in apple

Abiotic stress of plants has serious consequences on the development of the apple industry. Nuclear pore complexes (NPCs) control nucleoplasmic transport and play an important role in the regulation of plant abiotic stress response. However, the effects of NPCs on apple salt and osmotic stress responses have not been reported yet. In this study, we analyzed the expression and function of NUCLEOPORIN 62 (MdNup62), a component of apple NPC. MdNup62 expression was significantly increased by salt and mannitol (simulated osmotic stress) treatment. The MdNup62-overexpressing (OE) Arabidopsis and tomato lines exhibited significantly reduced salt stress tolerance, and MdNup62-OE Arabidopsis lines exhibited reduced osmotic stress tolerance. We further studied the function of HEAT SHOCK FACTOR A1d (MdHSFA1d), the interacting protein of MdNup62, in salt and osmotic stress tolerance. In contrast to MdNup62, MdHSFA1d-OE Arabidopsis lines showed significantly enhanced tolerance to salt and osmotic stress. Our findings suggest a possible interaction of MdNup62 with MdHSFA1d in the mediation of nuclear and cytoplasmic transport and the regulation of apple salt and osmotic stress tolerance. These results contribute to the understanding of the salt and osmotic stress response mechanism in apple.

function was the first and most widely studied in plants.Several plant HSFs are involved in the regulation of high temperature tolerance, such as HSFA1, HSFA2, HSFA9, HSFB1, and HSFB2 [18][19][20][21] .Additionally, HSFs are also involved in drought and salt stress responses.Under drought stress, the HEAT SHOCK PROTEIN 90 (MdHSP90)-MdHSFA8a complex dissociates to release MdHSFA8a, which further interacts with RELATED TO AP2 12 (RAP2.12)to activate downstream gene activity and enhance plant survival 22 .The drought-and salt-tolerance of OsHSFB2b overexpressing plants decreased significantly, whereas the tolerance of OsHSFB2b-RNAi lines increased significantly 23 .Arabidopsis AtHSFA6a is also involved in the regulation of salt-tolerance and drought-tolerance pathways 24 .
Apple (Malus × domestica Borkh.) is widely cultivated in temperate regions of the world and is an economically important fruit tree.However, due to global climate change, salt stress presents serious challenges to the development of the apple industry.Therefore, it is important to strengthen the research on related molecular mechanisms.To date, there have been few reports on the molecular mechanism of salt-tolerance in apple.Genes, such as C-REPEAT BINDING FACTORS (MdCBF), AUTOPHAGY-RELATED 10 (MdATG10), Na + -H + EXCHANGER 1 (MdNHX1), and ETHYLENE RESPONSE FACTORS 3/4 (MdERF3/4) are involved in the regulation of salt stress response in apple [25][26][27][28] .However, the molecular regulatory network in response to salt stress is relatively complex, and the research on apple is underdeveloped and difficult to conduct.Further research on salt tolerance of apple is required.
Our previous studies show apple MdNup62 interacts with several MdHSFAs and participates in the heat stress response pathway 29 .In the present study, we found that apple MdNup62 and MdHSFA1d were significantly induced by salt and mannitol treatment.And the salt and osmotic stress tolerance of MdNup62-OE plants were significantly reduced, whereas the MdHSFA1d-OE plants were significantly increased.These findings suggest that both MdNup62 and MdHSFA1d are involved in the regulation of salt and osmotic stress responses in apple.

Plant materials and growth conditions
'Nagafu No. 2′ plants were grown on MS medium containing 0.1 mg L −1 indolebutyric acid and 0.6 mg L −1 6-benzylaminopurine under long-day conditions (16 h-light/8 h-dark) at 24 °C and were sub-cultured every 45 d.Arabidopsis plants ('Columbia') were grown on 1/2MS medium under long-day conditions (16 h-light/8 h-dark) at 22 °C.And the components of 1/2MS plates are MS 2.2 g/L, Agar 7 g/L, Sucrose 30 g/L.Tomato plants (' Ailsa Craig') were grown on MS medium under long-day conditions (16 h-light/8 h-dark) at 25 °C.And the components of MS plates are MS 4.4 g/L, Agar 7 g/L, Sucrose 30 g/L.Arabidopsis and tomato seeds were previously preserved in our laboratory.

Protein alignment and phylogenetic relationship analysis
We downloaded HSFA1d protein sequences of nine plant species (Arabidopsis, Malus domestica, Populus trichocarpa, Oryza sativa, Rosa chinensis, Pyrus bretschneideri, Ananas comosus, Prunus dulcis, and Glycine max) from NCBI.And a protein sequence alignment of these nine plant species was performed using DNAMAN software.A phylogenetic tree comprising HSFA1d from nine species was constructed using the MEGA-X program.

RNA extraction and qRT-PCR analysis
Total RNA was extracted from apple and Arabidopsis seedlings using an RNA Plant Plus Reagent Kit (TIANGEN, Beijing, China).cDna was synthesized from the RNA using a PrimeScript RT Reagent Kit (Takara, Shiga, Japan).qRT-PCR analysis was conducted on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, USA).The reaction solution contained 10 μL of SYBR Green I Master Mix (CWBIO, Beijing, China), 0.5 μmol L −1 primers (SANGON BIOTECH, Shanghai, China) (Table S1), and 1 μL of each template, resulting in a total volume of 20 μL.The PCR program was: 95 °C for 3 min; 40 cycles of 94 °C for 15 s, 60 °C for 20 s, and 72 °C for 15 s.All the samples were analyzed with three biological replicates, each comprising three technical replicates.Relative gene expression levels were calculated in accordance with the 2 −ΔΔCt method 30 .

Genetic transformation
Genetic transformations were performed by agrobacterium infection methods in accordance with published methods for Arabidopsis 31 and tomato (' Ailsa Craig') 32 plants.The transgenic Arabidopsis and tomato lines were grown on MS plates supplemented with 50 mg L −1 and 100 mg L −1 kanamycin, respectively.

Salt-and mannitol-tolerance assays
The 'Fuji' plants at 30 d after propagation were used for the 150 mmol L −1 salt and 300 mmol L −1 mannitol treatment.We collected leaf samples before and at 1, 3, and 6 h after each of the two treatments.The samples were immediately frozen in liquid nitrogen and stored at − 80 °C.
The transgenic Arabidopsis seeds were grown in 1/2MS medium containing 75 and 100 mmol L −1 salt, and 100 and 200 mmol L −1 mannitol.The transgenic tomato seeds were grown in MS medium containing 100 mmol L −1 salt.On the 8th day after germination, we calculated the root length and the fresh weight of the seedlings.

Statistical analyses
Statistical analyses were performed using SPSS software.Data are reported as means ± SDs.Asterisks (*) indicate significant differences between treatments as assessed using Student's t-test at P < 0.05 (*) and P < 0.01 (**).Different lowercase letters above the bars indicate significant differences (P < 0.05, Tukey's test).

Expression analysis of MdNup62
We exposed apple tissue-cultured seedlings to salt and mannitol treatments.MdNup62 expression level was determined at different times during the salt and mannitol treatments (Fig. 1).MdNup62 was significantly induced by salt, and its expression level was highest at 3 d after salt treatment (Fig. 1B).MdNup62 was also significantly induced by mannitol; its expression level was highest at 3 d and 6 d after mannitol treatment (Fig. 1C).

Overexpression of MdNup62 reduces salt-stress tolerance
To confirm the role of MdNup62's in salt-stress tolerance, we performed an Agrobacterium-mediated genetic transformation of MdNup62 into Arabidopsis.We found that the root length of MdNup62-OE lines were significantly shorter and their fresh weight were lower than that of WT on MS culture medium with both 75 and 100 mM salt treatment, whereas there was no significant difference in root length and fresh weight without salt treatment (Fig. 2A-C).The presence of the transgene in MdNup62-OE lines was confirmed using qRT-PCR (Figure S1A).Because our previous research found that MdNup62 interacts with some MdHSFs 29 , and HSPs are located downstream of HSFs.So we performed a qRT-PCR analysis of four AtHSPs (AtHSP101, AtHSP70T-2, AtHSP22.0-ER, and AtHSP21) (Fig. 2D).Their expression levels in transgenic Arabidopsis were reduced under salt-stress conditions.Moreover, salt-stress tolerance assays were carried out in transgenic tomato plants (Fig. 3).As in transgenic Arabidopsis, the root length of transgenic tomato was significantly reduced compared with that of WT (Fig. 3A,B).The presence of the transgene in MdNup62-OE lines was confirmed by qRT-PCR (Fig. 3C).

MdNup62 overexpression reduces osmotic-stress tolerance
We studied the phenotype of MdNup62-OE lines in Arabidopsis under osmotic (mannitol) treatment and obtained same results to those of salt-stress treatment.The root length of MdNup62-OE lines were significantly shorter and their fresh weight were lower than that of WT on MS culture medium with both 100 and 200 mM mannitol treatment, whereas there was no significant difference in root length and fresh weight without mannitol treatment (Fig. 4A-C).And the expression levels of the four AtHSPs (AtHSP101, AtHSP70T-2, AtHSP22.0-ER, and AtHSP21) in transgenic Arabidopsis were reduced under osmotic-stress conditions (Fig. 4D).

Sequence and expression analysis of MdHSFA1d
Because our previous research found that MdNup62 interacts with MdHSFs 29 , we chose one of the interacting proteins, MdHSFA1d, to investigate its relationship with the function of MdNup62.And we performed the sequence analysis of MdHSFA1d first.Multiple sequence alignment of HSFA1d homologs of nine plant species (Arabidopsis, Malus domestica, Populus trichocarpa, Oryza sativa, Rosa chinensis, Pyrus bretschneideri, Ananas comosus, Prunus dulcis, and Glycine max) revealed a universally conserved HSF domain (Fig. 5A).Phylogenetic analysis indicated that MdHSFA1d was closely related to genes in Pyrus bretschneideri, Prunus dulcis, and Rosa chinensis in the family Rosaceae, but is more distantly related to genes in monocotyledons (Oryza sativa and Ananas comosus) (Fig. 5B).MdHSFA1d expression was determined at different times under the salt and mannitol treatments (Fig. 5C,D).MdHSFA1d was significantly induced by salt, and its expression level was highest at 3 d and 6 d after salt treatment.MdHSFA1d was also significantly induced by mannitol, with expression levels highest at 6 d after mannitol treatment.

MdHSFA1d overexpression promotes salt-stress tolerance
To verify the salt-stress tolerance phenotype of MdHSFA1d, we performed Agrobacterium-mediated genetic transformations of MdHSFA1d into Arabidopsis.The root length of MdHSFA1d-OE lines were significantly longer and their fresh weight were higher than that of WT on MS culture medium with both 75 and 100 mM salt treatment, whereas there was no significant difference in root length and fresh weight without salt treatment (Fig. 6A-C).The presence of the transgene in MdHSFA1d-OE lines was confirmed using qRT-PCR (Figure S1B).And a qRT-PCR analysis of four AtHSPs (AtHSP101, AtHSP70T-2, AtHSP22.0-ER, and AtHSP21) were also performed (Fig. 6D).Their expression levels in transgenic Arabidopsis were increased under salt-stress conditions.

MdHSFA1d overexpression promotes osmotic-stress tolerance
To study the osmotic-stress tolerance phenotypes of MdHSFA1d, we exposed MdHSFA1d-OE transgenic plants to osmotic-stress (mannitol).The root length of MdHSFA1d-OE lines were significantly longer and their fresh weight were higher than that of WT on MS culture medium with both 100 and 200 mM mannitol treatment, whereas there was no significant difference in root length and fresh weight without mannitol treatment (Fig. 7A-C).And the expression levels of four AtHSPs (AtHSP101, AtHSP70T-2, AtHSP22.0-ER, and AtHSP21) in transgenic Arabidopsis were increased under osmotic-stress conditions (Fig. 7D).

Discussion
Although the key factors of abiotic stress signaling in plants have been identified, the abiotic stress response is complex and several other factors have not been discovered yet.As channels of material communication between the cytoplasm and nucleus, nucleoporins are involved in the regulation of abiotic stress pathways in plants.Nucleoporin CPR5, HOS1, and Nup160 are involved in the temperature stress pathway 14,16,33 .The expression of ABA and salt-induced stress response genes, such as RESPONSIVE TO DESICCATION 29A (RD29A), COLD-REGULATED 47 (COR47), COR15A, was significantly decreased in nup85, nup160, and hos1 mutant lines.MEDIATOR SUBUNIT 18 (MED18) and Nup85 exhibit direct interaction with each other, and both MED18 and Nup85 mutants show increased sensitivity to ABA and salt stress with attenuated expression of stress response genes 15 .In our previous study, we found that overexpression of apple MdNup62 significantly reduced heat tolerance in Arabidopsis 29 .In the present study, MdNup62 was significantly induced by salt and mannitol www.nature.com/scientificreports/(osmotic simulation) treatments (Fig. 1).Moreover, the MdNup62-OE Arabidopsis lines exhibited significantly reduced salt and osmotic tolerance (Figs. 2, 4).Many HSP genes have been reported to be associated with saltand osmotic-stress.For example, HSP70, HSP40, HSP17.0, and HSP23.7 were positive regulating salt-stress tolerance [34][35][36] , while HSP70,and HSP90 were related to osmotic-stress tolerance 37,38 .And in this study, we found that four AtHSPs (AtHSP101, AtHSP70T-2, AtHSP22.0-ER, and AtHSP21) in MdNup62-OE Arabidopsis lines were reduced under salt-and osmotic-stress conditions (Figs. 2, 4).So, we speculate that MdNup62-OE Arabidopsis lines negatively regulate salt-and osmotic-stress tolerance by reducing the expression level of HSP genes.These results indicate that MdNup62 is a factor in salt and osmotic stress responses in apple.HSFs are involved in the regulation of several abiotic stress pathways.For example, Arabidopsis AtHSFA6a positively affects the regulation of drought and salt tolerance, whereas rice OsHSFB2b and maize ZmHSF08 exhibit negative regulatory effects 23,24,39 .Apple MdHSFA8 enhanced plant viability under drought conditions by promoting the accumulation of flavonoids and scavenging reactive oxygen species 22 .In our previous study, we found that both MdHSFA1d-OE and MdHSFA9b-OE Arabidopsis exhibited significantly increased heat tolerance 29 ; similar results were obtained in this study.MdHSFA1d was significantly induced by salt and mannitol (osmotic simulation) treatments (Fig. 5).When treated with salt and mannitol, the MdHSFA1d-OE Arabidopsis exhibited better growth (Figs.6, 7).The HSP genes are located downstream of the HSF transcription factors and are directly or indirectly regulated by them 40 .Unlike MdNup62-OE Arabidopsis lines, these four AtHSPs (AtHSP101, AtHSP70T-2, AtHSP22.0-ER, and AtHSP21) in MdHSFA1d-OE Arabidopsis lines were increased under salt-and osmotic-stress conditions (Figs. 6, 7).This may be an important reason for the phenotypic differences between the two transgenic lines.These results indicate that MdHSFA1d is also a factor in salt and osmotic stress responses in apple.
Apple MdNup62 and MdHSFA1d proteins interact directly with each other and present opposite phenotypes under high temperature stress 29 ; their overexpression in Arabidopsis also presents opposite phenotypes under salt and osmotic stress conditions.Therefore, we hypothesized that MdNup62, as a nucleoporin, may hinder HSF transport and negatively affect the regulation of salt and osmotic stress responses.However, previous studies found that both Nup62 deletion and overexpression strains of Arabidopsis exhibit increased sensitivity to auxin, indicating that overexpression does not result in functional gain, but rather functional loss 41 .With osmotic and salt treatments, apple MdHSFA1d was significantly induced and transported into the nucleus through NPC channels to promote downstream HSPs expression in WT, thereby enhancing osmotic and salt tolerance.In MdNup62-OE lines, the structure of the apple NPC changed, blocking the transport of MdHSFA1d into the nucleus and causing plant injury.Based on these results, we inferred that apple MdNup62 is involved in the regulation of salt and osmotic stress responses by controlling the transport of MdHSFA1d between the nucleus and cytoplasm.

Figure 1 .
Figure 1.Expresion analysis of MdNup62 under salt and mannitol treatment.(A) The phenotype of 'Nagafu No. 2' tissue-cultured seedlings at 0, 1, 3, and 6 h under salt(upper panels) and mannitol(lower panels) treatment conditions.Bar = 1 cm.(B) and (C) Analyses of MdNup62 expression levels in 'Nagafu No. 2' tissue-cultured seedling leaves at 0, 1, 3, and 6 h under (B) salt and (C) mannitol treatment conditions.Each sample was analysed with three biological replicates, each comprising three technical replicates.Means followed by different lowercase letters are significantly different at the 0.05 level.The same below.

Figure 2 .
Figure 2. MdNup62 reduced salt-stress tolerance in Arabidopsis.(A) Phenotype of the MdNup62overexpression Arabidopsis lines for salt-stress tolerance.Bar = 1 cm.(B) and (C) Statistical analysis of (B) primary root length and (C) fresh weight of WT and MdNup62-overexpression Arabidopsis lines after the salt treatment.Asterisks denote significant differences as determined by a t-test (*P < 0.05).(D) qRT-PCR analysis of four AtHSPs expression in Arabidopsis samples after 100 mM NaCl treatment.Asterisks denote significant differences as determined by a t-test (*P < 0.05).The same below.

Figure 3 .
Figure 3. MdNup62 reduced salt-stress tolerance in tomato.(A) Phenotype of the MdNup62-overexpression tomato lines for salt-stress tolerance.Bar = 1 cm.(B) Statistical analysis of primary root length of WT and MdNup62-overexpression tomato lines after the salt treatment.(C) qRT-PCR analysis of MdNup62 expression in tomato samples.

Figure 4 .
Figure 4. MdNup62 reduced osmotic-stress tolerance in Arabidopsis.(A) Phenotype of the MdNup62overexpression Arabidopsis lines for osmotic-stress tolerance.Bar = 1 cm.(B) and (C) Statistical analysis of (B) primary root length and (C) fresh weight of WT and MdNup62-overexpression Arabidopsis lines after the mannitol treatment.(D) qRT-PCR analysis of four AtHSPs expression in Arabidopsis samples after 200 mM mannitol treatment.

Figure 6 .
Figure 6.MdHSFA1d promotes salt-stress tolerance in Arabidopsis.(A) Phenotype of the MdHSFA1doverexpression Arabidopsis lines for salt-stress tolerance.Bar = 1 cm.(B) and (C) Statistical analysis of (B) primary root length and (C) fresh weight of WT and MdHSFA1d-overexpression Arabidopsis lines after the salt treatment.(D) qRT-PCR analysis of four AtHSPs expression in Arabidopsis samples after 100 mM NaCl treatment.

Figure 7 .
Figure 7. MdHSFA1d promotes osmotic-stress tolerance in Arabidopsis.(A) Phenotype of the MdHSFA1doverexpression Arabidopsis lines for osmotic-stress tolerance.Bar = 1 cm.(B) and (C) Statistical analysis of (B) primary root length and (C) fresh weight of WT and MdHSFA1d-overexpression Arabidopsis lines after the mannitol treatment.(D) qRT-PCR analysis of four AtHSPs expression in Arabidopsis samples after 200 mM mannitol treatment.
Prior to conducting the research, the permission from Hebei Normal University of Science and Technology and Horticulture College of Northwest A & F University to collect and analyse the Fuji, Arabidopsis, and Tomato sample documented in this work was obtained.All the experimental materials in this study do not violate the IUCN Policy Statement on Research Involving Species at Risk of Extinction and Convention on the Trade in Endangered Species of Wild Fauna and Flora, and have been approved by the government.